Antiobotic alpha - methyldethiobiotin alpha methylbiotin and their esters

ABSTRACT

ANTIBIOTICS A-METHYLDETHIOBIOTIN, A-METHYLBIOTIN, AND THEIR ESTERS, A-METHYLDETHIOBIOTIN AND ITS ESTERS HAVE THE FOLLOWING FORMULA:   4-CH3,5-(R-OOC-CH(-CH3)-(CH2)4-)IMIDAZOLIDIN-2-ONE   WHEREIN R IS HYDROGEN OR T-(BUTYL, PHTHALIMIDOMETHYL AND PHENACYL. A-METHYLBIOTIN AND ITS ESTERS HAVE THE FOLLOWING FORMULA:   4-(R-OOC-C(-CH3)-(CH2)3-)HEXAHYDROTHIENO(3,4-D)-   IMIDAZOL-2-ONE   WHEREIN R IS AS DEFINED ABOVE. A-METHYLDETHIOBIOTIN, WHERE R IS HYDROGEN IN FORMULA I, AND A-METHYLBIOTIN, WHERE R IS HYDROGEN IN FORMULA II, ARE PRODUCED IN A LYDIMYCIN FERMENTATION. THESE COMPOUNDS ARE ACTIVE AGAINST MYCOBACTERIUM AVIUM AND CAN BE USED TO CONTROL THIS MICROORGNISM WHICH IS A KNOWN PRODUCER OF GENERALIZED TUBERCULOSIS IN BIRDS AND RABBITS.

United States Patent Olfice 3,658,837 Patented Apr. 25, 1972 3,658,837ANTIOBOTIC a METHYLDETHIOBIOTIN, aMETHYLBIOTIN, AND THEIR ESTERSLadislav J. Hanka and David G. Martin, Kalamazoo, Mich., assignors toThe Upjohn Company, Kalamazoo, Mi h No Drawing. Continuation-impart ofapplication Ser. No. 736,278, June 12, 1968. This application May 19,1969, Ser. No. 826,008

Int. Cl. C07d 49/30 U.S. Cl. 260309.7 10 Claims ABSTRACT OF THEDISCLOSURE Antibiotics u-methyldethiobiotin, a-methylbiotin, and theiresters. a-Methyldethiobiotin and its esters have the following formula:

I wherein R is hydrogen or t butyl, phthalimidomethyl and phenacyl.

a-Methylbiotin and its esters have the following formula:

H i CH3 s 2 NTEO Racial-(m g I II wherein R is as defined above.

a-Mcthyldethiobiotin, where R is hydrogen in Formula -I, anda-methylbiotin, where R is hydrogen in Formula II, are produced in alydimycin fermentation. These compounds are active against Mycobacteriumavium and can be used to control this microorganism which is a knownproducer of generalized tuberculosis in birds and rabbits.

The invention described herein was made in the course of, or under, acontract with the Department of Health, Education, and Welfare.

CROSS REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of copending application Ser. No. 736,278, filed onJune 12, 1968, and now abandoned.

BRIEF SUMMARY OF THE INVENTION a-Methyldethiobiotin (U-28,559) is thetrivial name for 11,5 dimethyl 2 oxo 4 imidazolidinehexanoic acid.a-Methylbiotin is the trivial name for cis-tetrahydrou-methyl 2oxo-thieno[3,4-d]imidazoline 4 valeric acid. These are acidic compounds.u-Methyldethiobiotin is active against various Gram-positive andGram-negative microorganisms, for example, Escherichia coli,Mycobacterium avium, and Bacillus subtilis. a-Methyldethiobiotin alsohas antifungal activity, for example, it is active against Nocardiaasteroz'des, Blastomyces dermititidis, and Phialophora verrucosa.a-Methylbiotin is active against various Gram-positive microorganisms,for example, Bacillus subtilis, Bacillus cereus, Mycobacterium avium,and Mycobacterium phlei.

DETAILED DESCRIPTION The production of a-methyldethiobiotin anda-methylbiotin in a lydimycin fermentation, as disclosed in U.S. Pat.3,395,220 employs the microorganism Streptomyces lydicus, NRRL 2433.This microorganism is available to the public, without restriction, fromthe Northern Utilization Research and Development Division, AgriculturalResearch Service, U .S. Department of Agriculture, Peoria, 111., USA.a-Methyldethiobiotin and a-methylbiotin also can be produced by anotherstrain of S. lydicus having the repository number NRRL 3432. Asubculture of this strain also is available to the public, withoutrestriction, from the above-mentioned repository.

a-Methyldethiobiotin and a-methylbiotin are detected in the lydimycinfermentation liquor by a new procedure which involvespaper-chromatographing the fermentation liquor in a Sarm'a 1 system. Thedeveloped papergram is bioautogra-phed against Bacillus swbtilis grownin a synthetic agar medium. The Sarina 1 system consists of a papergramwhich is developed with the upper phase of a solvent mixture consistingof l-butanol, methanol, benzene, and water *(2:1:1:1). The syntheticmedium upon which Bacillus subtilis is grown consists of the following:

G. Na HPO -7H O 1.7 KH PQ, 2.0 02 4 MgSO 0.1 Glucose 2.0 B-acto agar 115.0

Distilled water1 liter. Metallic ion stock solution 1 ml.

1 Bacto agar provided by Difco Laboratories, Detroit, Mich. Metallic ionstock solution consists of the following:

Meg. ml. NaMOOs-2Ha0 200 CoClz Cu S04 100 Mn S04 2 CaCla 25 FeCla -4H2O5 ZnCla ZnCla has to be dissolved separately using a drop of 0.? NHCIfor 10 ml. of water. The stock solution is heated to bring all thecompounds in solution, kept standing for 24 hours, and sterile filtered.

The above-disclosed procedure for detecting a-methylde thiobiotin anda-methylbiotin in a lydimycin fermentation beer was made availablesubsequent to the lydimycin invention. Thus, u-methyldethiohiotin anda-methylbiotin present in lydimycin fermentation liquors heretofore wasnot detected and, accordingly, not recovered or used.

a-Methyldethiobiotin and u-methylbiotin can be recovered from alydimycin fermentation broth by the use of surface active adsorbents,for example, decolorizing resins and activated carbon (preferred).Advantageously, the lydimycin whole fermentation broth is first filteredat pH 4, or centrifuged to separate the mycelial and undissolved solidsfrom the broth. The clear beer is then passed over an adsorbent, forexample, activated carbon. The column is washed with a beer volume ofdeionized water and then with three column volumes each of 25% methanolin water, 50% methanol, and methanol. u-Methyldethiobiotin,a-methylbiotin, and lydimycin are eluted off the column with 20-columnvolumes of about 1-5 triethylamine in methanol. The fractions areanalyzed by paper chromatography with the Sarina 1 papergram system,described above. Evaporation of the solvent from the eluates,

dissolution of the residues in water, and acidification to a pH of about2.0 affords a crystalline mixture of a-methyldethiobiotin,u-methylbiotin, and lydimycin. Recovery of the individual antibioticsfrom this antibiotic mixture is readily accomplished by conversion ofthe antibiotic mixture to an ester form, preferably the phenacyl form.ther suitable esters are tertiary butyl, and phthalimidomethyl. Theantibiotic mixture ester is then recrystallized several times from asuitable solvent, for example, ethanol, acetone-ethanol, methylenechloride-ethanol, or methylene chloride-toluene to separate outlydimycin esters substantially free of the esters ofa-methyldethiobiotin and a-methylbiotin which remain in the motherliquors.

The mother liquors, containing predominantly the esters ofu-methyldethiobiotin and a-methylbiotin, and some lydimycin, arecombined and evaporated to a residue. This residue is extracted withtoluene saturated with propylene glycol and the extract is submitted topartition column chromatography. Fractions containing only the esters ofa-methyldethiobiotin and a-methylbiotin are subjected to furtherpurification over a silica gel chromatography column eluted with 3%ethanol in chloroform to give fractions containing a-methyldethiobiotinester substantially free of wmethylbiotin ester, and a-methylbiotinester substantially free of u-methyldethiobiotin ester.

The active fractions from the partition chromatography and silica gelchromatography are ascertained by either paper chromatography using B.subtilis grown in a synthetic medium, as described previously, or bythin-layer chromatography. This latter test is preferred. It isperformed by spotting test samples on silica gel coated glass slidesdeveloped with 5% methanol and chloroform. The active zones arevisualized by ultra-violet light. Lydimycin is the most polar, anda-methyldethiobiotin is the least polar, with a-methylbiotin having anintermediate polarity.

The ester group from esters of u-methyldethiobiotin and u-methylbiotincan be removed, advantageously, with a base, for example, sodiumthiophenoxide, sodium phenoxide, ammonia, and sodium hydroxide, toafford crystalline a-methyldethiobiotin and ot-methylbiotin,respectively. These crystals can be purified further byrecrystallization from acetone.

The above method for purifying a-methyldethiobiotin and a-methylbiotinis especially good for separating these antibiotics from materialcontaining a-methyldethiobiotin, a-methylbiotin, and lydimycin since theesters of a-rnethyldethiobiotin and a-methylbiotin are cleanly isolatedby chromatography on a silica gel column.

a-Methyldethiobiotin also can be recovered from a lydimycin fermentationbroth by filtering the whole broth at pH 4, passing the filtrate overactivated carbon, eluting the activated carbon with 3-column volumeseach of methanol in water, 50% methanol, and substantially anhydrousmethanol. This elution is then followed by an elution of the column withabout ZO-column volumes of 5% triethylamine in methanol. The eluatefractions containing u-methyldethiobiotin substantially free oflydimycin can be concentrated to asolid material and purified by silicagel chromatography.

Alternatively, the solid material containing a-methyldethiobiotinsubstantially free of lydimycin can be purified by first dissolving thesolid material in ammonium hydroxide, filtering the solution, and thenacidifying the solution to a pH of about 2.0 to yield essentially puretit-methyldethiobiotin crystals. Recrystallization from acetone resultsin further purification of the a-methyldethiobiotin crystals.

Salts of u-methyldethiobiotin and m-methylbiotin are formed employingthe free acid of these compounds and an inorganic or organic base. Thesesalts can be prepared, for example, by dissolving a-methyldethiobiotinor mmethylbiotin free acid in an alcohol, for example, methanol andethanol, adding a dilute base until the pH of the solution is about 10.0to 11.0, and concentrating and drying the solution to provide a driedresidue consisting of the base salt. Salts which can be formed includethe sodium, potassium, and calcium salts. Other salts, including thosewith organic bases such as primary, secondary, and tertiary monoamines,as well as the polyamines, also can be formed using the above-describedor other commonly employed procedures. Other valuable salts are obtainedWith therapeutically eifective bases which impart additionaltherapeutical efiiects thereto. Such bases are, for example, the purinebases such as theophyllin, theobromin, caffeine, or derivatives of suchpurine bases; antihistaminic bases which are capable of forming saltswith weak acids; pyridine compounds such as nicotinic acid amide,isonicotinic acid hydrazide, and the like; phenylalkyl amines such asadrenaline, ephredrine, and the like; choline, and others. Salts ofa-methyldethiobiotin and a-methylbiotin can be used for the samebiological purposes as the free acid.

-Methyldethiobiotin and u-methylbiotin have an antibacterial spectrum asshown in the following tables. The antibacterial spectrum was determinedby a standard disc plate agar diffusion test using 13 mm. size paperdiscs.

The microorganisms were grown in the following media:

B. subtilis: Streptomycin assay agar-BBL S. lutea: Seed agar-BBL E.coli: Nutrient agarBBL S. aureus: Nutrient agarBBL M. avium: Brain heartinfusion agar-Difco The synthetic media had the following composition:

NazHPOqYHzO, g...

Agar, g Distilled water, 1 Metallic ion Stock solution mlompounConcentration -mcg./ml 200 CoClz mcg./ml CUSO4 mcg./ml 100 MnSOi mg./mli. CaClz mg./ml 25 FGC12'4H2O mg./ml 5 ZDCle 1 .mg./ml 5 ZnCl2 has to bedissolved separately using a drop of 0.1 N HCl for 10 ml. of water.

The stock solution is heated to bring all the compounds in solution,kept standing for 24 hours and sterile filtered.

Antibacterial activity of a-methy'ldethiobiotin Zone size (in mm.) at aTest organism: concentration of 1 mg./ml.

Bacillus subtilis 0 Bacillus subtilis (synthetic medium) 67 Sarcinalutea 0 Escherichia coli (hazy) 18 Escherichia coli (synthetic medium)22 Staphplococcus aureus O Mycobacterium avium 33 Antibacterial activityof a-methylbiotin Zone size (in mm.) at a concen- Test organism: trationof 686 meg/ml.

Bacillus cereus 24 Bacillus subtilis (grown in nutrient agar) 17Bacillus subtilis (grown in synthetic agar) 70 Mycobacterium avium 46Mycobacterium phlei 35 Antifungal activity of a-mezhyldethiobiotinMinimum inhibitory concen- Test organism: tration in meg/ml.

Nocardia asteroides 500 Blastomyces dermatitidis 500 Plzialophoraverrucosa 500 The new compounds of the invention, a-methyldethio biotinand a-methylbiotin, can be used in birds and rabbits to control theorganism Mycobacterium avium which is a known producer of generalizedtuberculosis in these animals. They also can be used in petroleumproduct storage to control the microorganism Bacillus subtilis which isa known slime and corrosion producer in petroleum products storage.a-Methyldethiobiotin also can be used to inhibit the microorganismEscherichia call which is a known odor producer and possible source oftrouble in papermill systems.

The novel phenylacyl esters of a-methyldethiobiotin and a-methylbiotinare active against Mycobacterium avium and Bacillus subtilis. Thus,these compounds can be used to inhibit these microorganisms. Further,the esters of amethyldethiobiotin and ot-methylbiotin are useful in apurification process, as described herein, to separate lX-methyldethiobiotin and a-methylbiotin from lydimycin.

The following examples are illustrative of the process and products ofthe present invention but are not to be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1.-a-METHYLDETHIOBIOTIN Part A.Fermentatin A soil stock ofStreptomyces lydicus, NRRL 3432, is used to inoculate a series ofl-liter Erlenmeyer flasks each containing 250 m1. of a sterile preseedmedium consisting of the following ingredients:

G./liter Glucose monohydrate 25 Pharmamedia 1 25 Tap water, q.s., 1liter.

Pharrnamedia is an industrial grade of cottonseed flour produced byTraders Oil Mill Company, Fort Worth, Tex.

The preseed inoculum is grown for 45 to 50 hours at 28 C. on areciprocating shaker.

Preseed inoculum, as described above, is used to inoculate a 3800 literseed tank containing 1250 liters of the following sterile seed medium:

Glucose monohydrate g./liter 25 Pharmamedia do 25 Ucon LB-625 1ml./liter 0.4

Tap water, q.s., 1 liter.

Ucon LB-625 is an antifoaming agent supplied by Union CarbideCorporation.

The pH of the medium is adjusted to 7.2 with 50% ammonium hydroxidebefore sterilization.

The seed tank is maintained at a temperature of 28 C. for 45 to 50 hourswith an aeration rate of 50 s.c.f.m.

6 (standard cubic feet per minute) and agitation at the rate of 150r.p.m.

The seed inoculum, described above, is used to inoculate a 15,000 gallonfermentation tank containing 50,000

liters of the following sterile fermentation medium:

Glucose monohydrate g./liter 10 Dextrin do 30 Pabsts yeast do 10Cottonseed meal do 10 Ucon LB625 ml./liter 0.2 Tap water, q.s., 1 liter.

1 Brewers yeast obtained from the Pabst Brewing Company.

The pH of the medium is adjusted to 7.2 with 50% sodium hydroxide beforesterilization. The Ucon LB-6 25 is added after the pH adjustment. Thefermentation tank is maintained at a temperature of 35 C. for about 94hours with an air fiow rate of about 800 s.c.f.m. and agitation at arate of about 83 r.p.m.

Part B.--Recovery (1) Whole beer (46,200 liters) from a fermentation, asdescribed above, is filtered at pH 4 with the aid of diatomaceous earth.The clear beer is adsorbed on 545 kg. of degassed carbon (PittsburghChemicals CAL carbon) and eluted with 9,000 liters of acetone; 600-literfractions are collected. The fractions are spotted against a tray of theassay microorganism Saccharomyces pastorianus to determine the activefractions. The active fractions (7,800 liters) are stripped of acetoneand adsorbed on 200 lbs. of degassed CAL carbon. The carbon is washedwith water and then gradiently eluted with 3200 liters of 50% toacetone. The first 420 liters are concentrated to 100 liters andadsorbed on 10 kg. of degassed CAL carbon and then gradiently elutedwith 260 liters of 50% to 100% acetone. The first 40 liters areconcentrated to 4.5 liters of a viscous solution containing 3104 g. ofsolids assaying 1 biounit/mg. against S. pastarianus. (A biounit isdefined as the concentration of the antibiotic which gives a 20 mm. zoneof inhibition under the standard assay conditions. Thus, if for examplea fermentation beer has to be diluted to give the 20 mm. zone ofinhibition, the potency of such beer is 100 Bu. per ml.) The next 20liters are concentrated to 4 liters of solution containing 548 g. ofsolids assaying 3 biounits/mg. against S. pastorianus. These twoconcentrates are stored under refrigeration after which a solidseparates from each concentrate. The first more viscous concentrate isdiluted with 3 volumes of water, chilled overnight, and filtered. Thecollected solid is washed with water and dried; yield, 10.96 g. of crudesolid rich in u-methyldethiobiotin. The presence of a-methyldethiobiotinis shown by paper chromatography with the Sarina No. 1 system, describedabove. The second concentrate, described above, afforded l5.25 g. of asolid rich in a-methyldethiobiotin.

(2) a-Methyldethiobiotin also can be recovered from the fermentationbeer described in Part A by the following recovery process. The wholefermentation beer is filtered at a pH of about 4.0 with the aid ofdiatomaceous earth. The clear beer is adsorbed on 20% beer volume ofdegassed CAL carbon in a chromatographic column. The column is washedwith a beer volume of deionized Water and then with 3-column volumeseach of 25% methanol in water, 50% methanol, and methanol.a-Methyldethiobiotin is eluted from the column, along with any lydimycinwhich may be present, with 20-column volumes of 5% triethylamine inmethanol. The eluate fractions are analyzed by paper chromatography withthe Sarma No. 1 system.

Part C.Purificati0n A portion of the crude solids containinga-methyldethiobiotin (6.3 g.), obtained in Part B, is dissolved in 50ml. of the upper phase obtained by mixing butanolzmethanolzbenzenezwater(2:1:1:1) and adsorbed onto a column prepared by slurrying 600 g. ofsilica gel (No. 7734 E. Merck AG., Darmstadt), in the same solventmixture. The column is eluted with the same solvent mixture and 20 ml.fractions are collected. Fractions containing OL-Il'lflthyldethiobiotinare detected by dipping paper discs in every other fraction, drying thediscs in a fume hood for 2 hours, and applying them to a tray ofBacillus subtilis (synthetic medium). Activity against Bacillus swblilzsdiscloses the presence of a-methyldethiobiotin. Fractions containinga-methyldethiobiotin, as determined by the above Bacillus subtilis assayprocedure, are evaporated under reduced pressure; yield, 5.71 g. ofresidue. This residue is triturated with water adjusted to pH 2.5;yield, 4.54 g. of crystalline a-methyldethiobiotin having a meltingpoint of 160-161 C. The crystals of wmethyldethiobiotin are dissolved inrefluxing acetone and the solution clarified with diatomaceous earth.The filtrate is concentrated; yield, 3.37 g. of a-methyldethiobiotincrystals having a melting point of 161-162 C. Upon recrystallization ofthese crystals from acetone there is obtained 3.17 g. of crystallineu-methyldethiobiotin having a melting point of 161.5-162.5 C.

An alternate procedure for purifying a-methyldethiobiotin from thesolids obtained in Part B is as follows. Crude solid as prepared in PartB (10.96 g.) is dissolved in 50 ml. of l N NH OH and filtered with theaid of diatomaceous earth. The filtrate is acidified with hydrochloricacid to a pH of about 2.0 and crystals of a-methyldethiobiotin form;yield, 9.34 g. of crystalline a-methyldethiobiotin having a meltingpoint of 1605-1615 C. These crystals of u-methyldethiobiotin arerecrystallized from acetone and then isopropanol; yield, 7.30 g. ofcrystalline a-methyldethiobiotin having a melting point of 161-162 C.

EXAMPLE 2 Eluate fractions containing ot-methyldethiobiotin, obtained asdescribed in Part B(2), are evaporated to dryness and dissolved in aminimum volume of water required to effect solution. The solution isadjusted to a pH of 2.0 with sulfuric acid and chilled for about 4 days.The resulting solid is collected, washed with cold water and dried. Thissolid is dissolved in dimethylformamide (19 mL/g.) and triethylamine(1.2 ml./g.) and treated with an equal weight of phenacyl bromide atroom temperature for about 1-2 hours. (This reaction can be carried outat C. for longer periods of time or at higher temperatures up to 60 C.for a shorter period of time. At least one equivalent or a slight excessof phenacyl bromide can be used. One equivalent of triethylamine or anexcess also can be used.) The resulting solution is poured into 20volumes of ice cold water and the resulting solid is collected, washedwith water and dried. This solid is dissolved in chloroform andchromatographed on silica gel (100 g./g.; E. Merck Ag, Darmstadt, No.7734). Elution of the silica gel column with 5% methanol in chloroformyields the phenacyl ester of a-methyldethiobiotin first and then thephenacyl ester of lydimycin. After recrystallization from acetone, thephenacyl ester of u-rnethyldethiobiotin, melting point 128-129 C., ishydrolyzed in the following manner. ml. of 1 N NaOH is added to asolution of 1.26 g. of the phenacyl ester of a-methyldethiobiotin in 200ml. of refluxing isopropanol. After 10 minutes at reflux, the solutionis cooled and evaporated to a residue. The residue is dissolved inwater, clarified with diatomaceous earth, and then acidified to a pH ofabout 2.0 with hydrochloric acid. The solid which forms is collected,washed with cold Water, dried, and recrystallized from acetone; yield,0.63 g. of a-methyldethiobiotin crystals having a melting point of160.5-162 C. (The hydrolysis of the ester group also can be carried outat room temperature by stirring a solution of the phenacyl ester ofa-methyldethiobiotin in tetrahydroiuran with 1 N NaOH for about 4 days.)

EXAMPLE 3 Upon substituting N-chloromethylphthalimide for phenacylbromide in Example 2, there is obtained the phthalimidomethyl ester ofa-methyldethiobiotin. This ester is readily cleaved at room temperaturewith anhydrous hydrogen chloride in ethyl acetate or dioxane solution,with hydrogen bromide in acetic acid, or with hydrazine hydrate inethanol to yield a-methyldethiobiotin.

Alternatively, :the tertiary butyl ester of u-methyldethiobiotin can beprepared by reacting its acid chloride with tertiary butyl alcohol andtriethylamine at 0 C. The tertiary butyl ester can be easily cleaved atroom temperature with hydrogen chloride in dioxane or with sulfuric acidto yield u-methyldethio'biotin.

EXAMPLE 4 Lydimycin, :x-methylbiotin, and oi-methyldethiob'iotin Wholebeer (46,200 liters) from a fermentation, as described in Example 1,Part A, is adjusted to pH 4.0 with sulfuric acid and filtered with theaid of diatomaceous earth. The filter cake is Washed with 0.1beer-volume of water and the cake is discarded. The clear beer isadsorbed on 545 kg. of degassed carbon (Pittsburgh Chemicals CAiLcarbon) and eluted with 9000 liters of acetone; 600- liter fractions arecollected. The fractions are spotted against a tray of the assaymicroorganism Saccharomyces pastorianus to determine thee activefractions.

The active fractions (7800 l.) are stripped of acetone and adsorbed on200 lbs. of degassed CAL carbon. The carbon is washed with water andthen gradiently eluted with 3200 liters of 50% to acetone. The first 420liters are processed for a-methyldethi0biotin as described in Example 1,Part B. The following eluates are pooled and concentrated to an aqueoussolution which is freeze dried atiording 8712 g. of residue (3.3 Bu./mg.against S. pastorianus). This residue is dissolved in 5 l. of the lowerphase of a mixture of ethylacetatetcyclohexane: Mcllvaines pH 3 bulier(Handbook of Chemistry and Physics, 22nd Edition, Chemical RubberPublishing Co., p. 960) in the proportions 16:4:1 and homogenized with10 kg. of Dicalite 4200 (a diatomaceous earth produced by Great LakesCarbon Corp., New York, NY.) and upper phase. The mixture is placed ontop of a column containing 52 kgm. of Dicalite 4200 which had beenslurried in upper phase with 20.8 1. of lower phase and the column iseluted with 1600 l. of upper phase. Concentration of the activefractions (S. pastorianws assay) to an aqueous solution and chillingaifords 15.9 g. of impure lydimycin assaying 2000-2260 Bu./mg. againstS. puszorianus, and 8.3 g. of less pure lydimycin assaying 5S5 Bu./mg.Rechromatography of the mother liquor residue with the same solventsystem alfords an additional 7.3 g. of lydimycin assaying 2000 Bu./mg.against S. pastorianus.

Paper strip analysis (Sarma 1 system) of these solids indicates twomajor zones of approximately equal size. These solids are extracted withan excess of l N NH OH. After clarification with diatomaceous earth, theextract is acidified to a pH of about 2.0 with hydrochloric acid; yield,24.3 g. of lydimycin which shows two major zones of activity on theSarma 1 system. This 24.3 g. of crude lydmycin is dissolved in asolution of 490 ml. of dimethylformamide and 29.2 ml. of triethylamine.After adding 22.08 g. of phenacyl bromide, the solution is stirred atroom temperature for 1.5 hours. The reaction mixture is then poured into9.8 liters of ice water. The resulting solid is collected, washed withwater, then with Skellysolve B (isomeric hexanes), and dried; yield,25.53 g. of crude lydimycin phenacyl ester. Paper strip analysis of thismaterial using a sheet saturated with 1:1 methanol: propylene glycol,and dried at 37 C. for 10 minutes followed by development With toluenesaturated with propylene glycol, shows two zones of activity against B.subtilis grown on synthetic agar. These zones show the presence oflydimycin phenacyl ester and a-methylbiotin phenacyl ester.

The crude lydimycin phenacyl ester is recrystallized three times fromethanol and three times from methylene chloride-toluene (the material isdissolved in refluxing methylene chloride and the methylene chloride isdisplaced with toluene) atfording 19.54 g., of pure lydimycin phenacylester crystals. The phenacyl ester is removed from the toluene insolublelydimycin phenacyl ester by the following procedure. A solution of 1.800g. of the phenacyl ester in 1 l. of tetrahydrofuran is stirred under anatmosphere of nitrogen 'with ml. of 1 N sodium hydroxide for 24 hr. atroom temperature. The solid which separates is collected, washed withacetone, and dissolved in water (approximately 100 ml.). The aqueoussolution is filtered and acidified affording 1.029 g. (85%) of lydimycinassaying 16,000 Bu./mg. against S. pastorianus, M.P. 251.5252 C.

Mother liquor residues from the recrystallizations, described above, areextracted with toluene saturated with propylene glycol and the extract(1.41 g.) is submitted to partition chromatography prepared as follows:Toluene (5 liters) and propylene glycol (600 ml.) are agitated and thelayers separated. Diatomaceous earth (1200 g.) is suspended in the upperphase, stirred for 30 minutes with 400 ml. of the lower phase, and thenpoured into a chromatographic column. The extract, described above, isdrained onto the column which is then eluted with l. of upper phasewhile collecting 50 ml. fractions. Active fractions are determined byapplying paper discs saturated with the test fraction to a tray of B.subrilis (grown in synthetic agar) and then analyzed by paperchromatography, as described above, and thin-layer chromatography (5%methanol in chloroform on silica gel detected by U.V.). Fraction 210shows a larger B. subtilis zone moving with a-methylbiotin than the zonemoving with amethyldethiobiotin but the U.V. analysis indicates thata-methyldethiobiotin is the major material present. (oc-Methyldethiobiotin is less polar than a-methylbiotin.)

Evaporation of fractions 21 0-258 leaves 70 mg. of residue which ischromatographed on 30 g. of silica gel (Merck, Darmstadt, No. 7734). Thecolumn is eluted with 3% ethanol in chloroform and ml. fractions arecollected and spotted against B. subtilis grown on syn thetic agar, asdescribed above. Again, active fractions are analyzed by paper stripchromatography using the microorganism B. subtilis (synthetic medium),and thinlayer chromatography, as described above. Fractions 2432 consistof essentially pure a-methylbiotin phenacyl ester.

A solution of a-methylbotin phenacyl ester (3 mg.) in methanol (3 ml.)is treated with 0.02 ml. of 1 N NaOH under an atmosphere of nitrogen.The solution is kept at room temperature for minutes and then warmed at50 C. for minutes. The methanol is evaporated and the residue isdissolved in 3 ml. of water and a paper chromatogram of the filtrate(Sarma 1 system) shows the presence of a-methylbiotin.

Characterization of u-methyldethiobiotin Crystals: White Titration: pKa'4.65; eq. wt. 233

Elemental analysis.Calcd for C H N O (percent): C, 57.87; H, 8.83; N,12.27. Found (percent): C, 58.02, 57.99; H, 8.70, 8.78; N, 12.41, 12.18.

Molecular weight: Calcd: 228.1474. Determined: 228.1472 (high resolutionmass spectrometer) Optical rotation:

l ltt=- (c, 0.98 in 95% EtOH).

Solubility: a-Methyldethiobiotin is very soluble in methanol, ethanol,aqueous ammonium hydroxide and alkali; it is soluble in isopropanol andacetone; it is relatively insoluble in Skellysolve B (isomeric hexanes)cyclohexane, ethyl acetate, methylene chloride, and benzene.

a-Methylbiotin is characterized, advantageously, as its phenacyl ester.Mass spectroscopy of the phenacyl ester of u-methylbiotin shows ameasured mass of 376.1456.

10 The theoretical is 376.1456 for C H N O S. Mass spectroscopy of thephenacyl ester of a-methylbiotin also shows a peak at 257.0949. Thiscorresponds to the theoretical of 257.0959 for a-methylbiotin u t'z a aOptical Rotary Dispersion (0RD) and NMR show that the methyl ina-methylbiotin is or to the carhoxyl group.

We claim: 1. A compound, u-methyldethiobiotin, which is substantiallyfree from lydimycin, and having the following structural formula:

or its pharmaceutically acceptable salts.

2. A compound as defined in claim 1, tar-methyldethiobiotin, in itscrystalline form.

3. A compound selected from the group consisting ofu-methyldethiobiotin, according to claim 1, or pharmaceuticallyacceptable salts thereof with alkali metals, alkaline earth metals, andamines selected from the group consisting of primary, secondary, andtertiary monoamines, and polyamines.

4. A compound selected from the group consisting of the t-butyl,phthalimidomethyl, and phenacyl esters of (it-methyldethiobiotin.

5. Phenacyl ester of u-methyldethiobiotin according to claim 4.

6. A compound, a-methylbiotin, which is substantially free fromlydimycin, and having the following structural formula:

II or its pharmaceutically acceptable salts.

7. A compound as defined in claim 6, rx-methylbiotin, in its crystallineform.

8. A compound selected from the group consisting of a-methylbiotin,according to claim 6, or pharmaceutically acceptable salts thereof withalkali metals, alkaline earth metals, and amines selected from the groupconsisting of primary, secondary, and tertiary monoamines, andpolyamines.

9. A compound selected from the group consisting of t-butyl,phthalimidomethyl, and phenacyl esters of umethyldethiobiotin.

10. Phenacyl ester of a-methylbiotin, according to claim 9.

References Cited UNITED STATES PATENTS 2,466,004 4/1949 Cheney et al260309.7 2,489,236 11/ 1949 Goldberg et al. 260309.7 2,492,373 12/ 1949Wood et a1. 260309.7 2,538,096 1/1951 Harris et al. 260309.7 3,395,2207/1968 Bergy et al. 424-417 NATALIE TROUSOF, Primary Examiner US. Cl.X.R. 195- 424'273 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTIONPatent No. 5, 5 57 Dated Apr i i 25, 972

Inventor) Lad is iav J Hanka and David G Martin It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

Coi umn 2, i ines 42 W, For Mcg./mi. read 200 mcg ./mi

200 100 mcg ./mi 100 100 mcg./mi. 100 2 mg./mi 2 25 mg./mi 25 5 mg ./mi

5 5 mg ./m] 5 Column 5, i ine 57, For "asoi id read a 50] id Column i,line 65, for-"Escherichia coi i (hazy) 18" read Escherichia coi i l8(hazy)- Column 8, i ine 2%, for "thee" read the line 59, For "iydmycin"read iydimycin Column 9, i ine 69, for "and a] kai i read and aqueous aikai i Signed and sealed this 31st day of October 1972.

(SEAL) Attestz EDWARD M.FLETCHER,JR. ROBERT GOT'I'SCHALK Attestinp;Officer Commissioner of Patents RM PO-105O (10-69) USCOMM-DC 60376-5 69& U.S. GOVERNMENT PRINTING OFFICE: I969 O366-334

